U N I V E R S 1 T A T I S MARIAE CURIE-SKŁODOWSKA LUBLIN — POLONIA
VOL. XXVI, 5 SECTIO C 1971
Instytut Mikrobiologii i Biochemii UMCS Zakład Biochemii
Jerzy ŁOBARZEWSKI
Isolation of Peroxidase from lnonotus radiatus Fungus
Izolacja enzymu peroksydazy z grzyba lnonotus radiatus
M3o/umMji 3H3MMa nepoKcnfla3bi M3 rpnóa lnonotus radiatus
INTRODUCTION
Investigations on fungal peroxidase carried out so far have mainly aimed to explain the physiological role of the enzyme. It is now recog- nized, on the grounds of the studies of Trojanowski and L e o n o- wicz (4, 15, 16, 17, 18, 19, 20), Ishikawa et al. (2, 3), and of L y r (5, 6, 7), that peroxidase and laccase co-operate in degradation processes of lignin molecule. The fungi grown under natural conditions i.e. on trees excrete oxidoreductases, namely laccase and peroxidase into the medium. A few years ago Ishikawa and his co-workers (2, 3) at- tempted to isolate peroxidase and laccase from Fomes fomentarius and Collybia uelutipes fungi. However, they did not succeed in isolating peroxidase from laccase. Reporting upon the properties of peroxidasc preparations being isolated Ishikawa (2) writes: ”The purified enzyme preparations, isolated from the mycelia and filtrates of both fungi, exhibited an absorption peak in the region of 410—440 nm on reaction with dithionite, thereby indicating the presence of peroxidase. Further- more, the preparations also exhibited a weak maximum at 405 nm, indi- cative of the presence of a heme-containing protein.”
Taking the above into consideration an attempt was madę to isolate peroxidase from lnonotus radiatus fungus which exretes peroxidase, as the only enzyme from oxidoreductase group, into the medium.
36 JerzyŁobarzewski MATERIAŁ AND METHODS
The filtrate of 3-week mycelium culture of Inonotus radiatus (Sow. Ex. Fr.
P. Karst H.M.J.P.C. No. 4335) was used for the isolation of peroxidase. The fungus mycelium was grown in the liquid medium which consisted in 3% malt extract. The medium was enriched with beech wood meal, which caused an intensive growth of the mycelium and an increased peroxidase biosynthesis (8).
The medium was prepared in 500 ml Roux flasks containing 150 ml of basie medium and 5 g of wood meal. After 3 weeks of fungus ineubation at 25°, the mycelium was filtered off and the filtrate was used as the starting materiał for the purification of peroxidase. Peroxidase from horseradish roots of Koch- -Light firm was also used in the experiments.
Peroxidase activity was determined by two methods, that of A u r a n d et al. (9) and of Seąueira et al. (13). p-phenylenediamine was the hydrogen donor in the former method while guaiacol in the latter. Protein content was estimated by the spectrophotometric method (11) and by the procedurę of Parnas-Wag- ner (11). The level of sugars bound with protein was assayed by the method of Wintzler (10).
RESULTS AND DISCUSSION
In the previous report (8) there were described the conditions of Inonotus radiatus growth in the liquid medium during which the amount of peroxidase synthetized by the fungus mycelium increased 30 times.
In the present experiments the filtrate after 3-week mycelium ineubation of Inonotus raditaus was used for isolation. The mycelium was discarded because, as compared with the filtrate, it contained only slightly larger amount of peroxidase but markedly larger amount of contaminating proteins, and because of the difficulties in its homogenization.
There were worked out four steps of the purification procedurę of peroxidase from the filrate. The filtrate was lyophilized and then dis- solved in a smali ąuantity of water. The solution was brought to 100%
ammonium sulfate saturation. After the dialysis of saturated peroxidase preparation the filtration was performed on various sephadexes. The best results of purification were obtained on G-100 sephadex. Column size, 2.5X45 cm (Fig. 1). 0.005 M phosphate buffer, pH=7 was used for elution. Under these conditions the 20-fold purification of peroxidase preparation was accomplished. Then, before being tranferred to a se- phadex column, the peroxidase solution was precipitated by ammounium sulfate for the second time. During precipitation the proteins of lower molecular weight were removed from the peroxidase preparation. During filtration through the sephadex the elution pattern changed in relation to the profile presented in Fig. 1 (Fig. 2). After filtration the 30-fold purification of peroxidase was achieved. Active fractions collected from 20 columns were lyophilized and stored at a Iow temperaturę. The
Fig. 1. Elution profile from G-100 sephadex chromatographic column.
Column size, 2.5 X 45 cm. Elution system: 0.005 M phosphate buffer,
pH = 7
Fig. 2. Elution profile from G-100 sephadex chromatographic column.
Column size, 2.5 X 45 cm. Elution system: 0.005 M phosphate buffer,
pH = 7
results of described steps of the preliminary purification of fungal pe- roxidase were listed in Table 1.
In order to characterize closer the obtained preparation of fungal peroxidase some analytical procedures were performed. Electrophoresis of the purified preparation of fungal peroxidase was carried out on polyacrylamide gel. The electrophoregram exhibited one main peroxi- dase band which corresponded to protein band on the gel submitted to parallel electrophoresis (electrophoretic conditions: Tris-glycine buffer, pH = 8.2, time 3 hrs., 3 mA). The second peroxidase fraction was faintly visible on gel. Therefore, fungal peroxidase shows smali heterogeneity in comparison, for instance, to horseradish peroxidase which, as S h a n- n o n et al. (14) reported, possesses 7 isozymes.
Then, there were marked the changes in protein content and there was estimated the content of sugars bound with protein during the succeeding steps of peroxidase purification (Table 2).
The influence of inhibitors on fungal peroxidase is analogous to that on horseradish peroxidase (12), which may suggest the similarity in the structure of both enzymes (Table 3). Argentum nitrate as a spe-
38 Jerzy Łobarzewski
Tab. 1. Comparison of the results of the purification of peroxidase enzyme of the filtrate of the culture of Inonotus radiatus fungus
Step of isolation
Volume Proteininmg CO*-»
c3
»-4►»"*
> 1
<=>
<o X Specificactivity Ratęof purification
sc IOo
W
C
£c oco
IM
W
4 tj
>>
uC
’o
«ł-tW
Lyophilisate of filtrate afterfungus
culture 2,000 39.0 1000.0 25.8 1.0 — 100
100% ammonium sulfate saturation 500 107.0 4000.0 37.5 1.5 — 100 2nd 100% ammonium sulfate satura-
tion 250 77.5 8000.0 103.0 4.0 0.175 97
Filtration on G-100 sephadex 160 9.7 7500.0 775.0 30.0 0.250 60
Tab. 2. Comparison of the resultsof protein assays according to P a r.n a s - W a g- ner method (11) and of sugars bound with protein by the method of Wintzler
(10) in the preparations of fungal peroxidase
Materiał % protein
per dry matter
mg% sugars bound with
protein
% sugar per 100mgprotein
Medium 5 176 61
Preparation after ammonium
sulfate treatment 90 70 5.1
Preparation after filtration on
G-100 sephadex 92 0.88 1.78
cifie inhibitor of flavine peroxidase was introduced into the experi- ments with inhibitors. According to Dollin’s report (1) flavine pe- roxidase is inhibited in 100% at AgNO3 concentration = 10-4 M. The results of the experiment with AgNO3 presented in Table 3 exclude a possibility of the presence of this kind of peroxidase in the examined materiał.
While examining the effect of pH upon the activity of fungal pero- xidase the comparison to horseradish peroxidase was madę (Fig. 3, 4).
Fig. 3. Effect of the concentration of hydrogen ions upon the activity of fungal and horseradish peroxida- ses. Guaiacol as hydrogen donor
Tab. 3. Influence of inhibitors on the
Fig. 4. Effect of the concentration of hydrogen ions upon the activity of fungal and horseradish peroxida- ses. p-phenylenediamine as hydrogen
donor
tivity of fungal peroxidase preparation
Inhibitor
Inhibitor concentration
in moles
Remaining peroxidase activity in%
% peroxidase inhibition
Hydroxylamine... 10~4 25 75
Hydroxylamine... 10-5 75 25
Hydroxylamine... 5X10-« 96 4
Thiourea... 10-* 68 32 Thiourea... 5X10-‘ 95 5 Sodium Diethyl-dithio-
carbamate... 10-s 0 100
Sodium Diethyl-dithio-
carbamate... 5X10-5 3 97
Sodium Diethyl-dithio-
carbamate... lO-’ 30 70
2,4-dichlorophenol .... 10-5 83 17
2,4-dichlorophenol .... 5X10-5 100 0
Sodium versenate .... 10-' 100 0
NaCN... 10-' 0 100 NaCN... 5X10-5 15 85 NaCN... lO-’ 25 75 NaCN... 5X10-’ 40 60 AgNO,... lO-4 100 0
40 Jerzy Łobarzewski
Guaiacol and p-phenylenediamine were used as hydrogen donors in those experiments. The preparations of fungal and horseradish pero- xidases had the identical maxima of reaction velocity depending upon pH of the medium.
Fig. 5. Effect of temperaturę upon the activity of fungal and horseradish peroxi- dases. Time of thermal inactivity, 15 min
During the investigation of the influence of temperaturę on the activity of fungal peroxidase there were shown the differences in the lability of the enzyme as compared with horseradish peroxidase (Fig. 5).
Then, there were plotted the absorption curves of purified fungal peroxidase in the visible region, and the diagram thus obtained was compared with that of horseradish peroxidase (Fig. 6). The preparations of purified fungal peroxidase were yellow and that was why the absorp
tion spectrum of the enzyme did not exhibit any maximum in the examined region.
On the basis of analogy between the action of horseradish peroxi- dase and that of fungal peroxidase it was expected that fungal peroxi- dase also consists of heme-containing protein which is characteristic of horseradish peroxidase. It was thought that the absorption maximum of fungal peroxidase in the Soret band is covered by unidentified yellow substances. An attempt was madę to remove the yellow colouring from the preparation of fungal peroxidase. For this purpose the absorption of peroxidase preparation was performed on active charcoal, aluminium oxide and hydroxyloapatite. The relatively best results were obtained by the use of active charcoal. At the same time there was observed a marked decrease in the peroxidase content of such a purified prepa
ration. But then the weak maximum was noted in the region of 400 nm, and after adding sodium dithionite there occurred the shift of maximum
Fig. 6. Absorption curves of fungal and horseradish peroxidases in the
visible region
Fig. 7. Absorption curves of fungal and horseradish peroxidases in the visible region. The changes in the absorption spectra under the influ
ence of sodium dithionite (Na2S2O4).
I — horseradish peroxidase, II — horseradish peroxidase after Na2S2O4 reduction, III — fungal peroxidase, IV —fungal peroxidase after Na2S2O4
reduction
to 430 nm (Fig. 7). Fungal peroxidase spectrum, as illustrated in Fig. 7, would be the evidence of hemoproteid structure of the enzyme.
The yellow colouring of purified preparation of fungal peroxidase may be attributed to the formation of enzyme-chinone complexes. The rise of such complexes seems to be indicated by the elution profile of fungal peroxidase on the sephadex, on which yellow substances together with peroxidase protein are eluted from the column.
The investigations on further steps of the purification of fungal peroxidase are continued.
The author wishes to express his thanks to Prof. Dr Jerzy Troja
nowski for his valuable remarks throughout this work.
42 Jerzy Łobarzewski
&
REFERENCES
1. Dolin M. J.: The Streptococcus faecalis Oxidases for Reduced Diphospho- pyridine Nucleotide. III. Isolation and Properties of Flavin Peroxidase for Reduced Diphosphopyridine Nucleotide. J. Biol. Chem., 225, 557 (1957).
2. Ishikawa H„ Schubert W. J., Nord F. F.: Investigation on Lignins and Lignification. Biochem Z., 338, 153 (1963).
3. Ishikawa H., Oki T.: The Oxidative Decomposition of Lignin. J. Japan.
Wood Res. Soc., 10, 207 (1964).
4. Leonowicz A., Trojanowski J.: Exoenzymes in Fungi Degrading Lignin. Acta Microbiol. Polon., 14, 55 (1965).
5. Lyr H.: Vorkommen von Peroxydase bei Holzzerstorenden Basidiomyceten.
Planta, 46, 408 (1955).
6. Lyr H.: Untersuchungen uber die Peroxydasen hóherer Filze. Planta, 48, 239 (1956).
7. Lyr H.: Uber den Nachweis von Oxydasen und Peroxydasen bei hóheren Pilzen und die Bedeutung dieser Enzyme fiir die Bavendamm-Reaction.
Planta, 50, 359 (1958).
8. Łobarzewski J.: Stimulation of Peroxidase Activity in Inonotus radiatus and Phellinus pini Fungi. Ann. Univ. Mariae Curie-Skłodowska, sectio C, 25, 15 (1970).
9. Methoden der enzymatischen Analyse. Red. B er g me y er H. U., Verlag Chemie G.M.B.H., Weinheim 1962.
10. Methods of Biochemical Analysis. Red. G1 ick D., Interscience Publishers, 2, New York 1955.
11. Methods in Enzymology. Red. Colo wiek S. P., Kapłan N. O., Academic Press, 3, New York 1957.
12. Saun der s B. C., H o 1 me s -Sied 1 e A. G., Stark B. P.: Peroxidase.
Wyd. Butterworth, London 1964.
13. Sequiera L., Mineo L.: Partial Purification and Kinetics of Indoleacetic Acid Oxidase from Tobacco Roots. Plant. Physiol., 46, 193 (1966).
14. Shannon L. M., Kay E., Lew J. Y.: Peroxidase Isozymes from Horsera- dish Roots. J. Biol. Chem., 241, 2166 (1966).
15. Trojanowski J.: Mikrobiologiczny rozkład ligniny. Post. Mikrobiol., 6, 151 (1967).
16. Trojanowski J.: Biological Degradation of Lignin. Int. Biodetn. Buli., 5, 119 (1969).
17. Trojanowski J., Leonowicz A.: Investigation on the Degradation of Lignin by Pholiota mutabilis. Ann. Univ. Mariae Curie-Skłodowska, sectio C, 18, 441 (1963).
18. Trojanowski J., Leonowicz A.: The Biodeterioration of Lignin by Fungi. Microbios., 3, 247 (1969).
19. Trojanowski J., Leonowicz A., Hampel B.: Exoenzymes in Fungi Degrading Lignin. Acta Microbiol. Polon., 15, 17 (1966)
20. Trojanowski J., Leonowicz A., Wojtaś M.: Exoenzymes in Fungi Degrading Lignin. Acta Microbiol. Polon., 15, 215 (1967).
STRESZCZENIE
Przeprowadzono próby oczyszczania peroksydazy zawartej w filtracie po 3-tygodniowych kulturach grzyba Inonotus radiatus. Pierwsze dwa stadia oczyszczania polegały na 2-krotnym strącaniu siarczanem amonu zagęszczonego przez liofilizację filtratu. Następnie zastosowano sączenie na sefadeksie G-100 (kolumna o wymiarach 2,5 X 45 cm). Podczas są
czenia na sefadeksie uzyskuje się 30-krotne oczyszczenie peroksydazy w stosunku do materiału wyjściowego. Oznaczono niektóre właściwości oczyszczonego preparatu peroksydazy grzybowej i porównano je z właś
ciwościami peroksydazy z chrzanu. Oznaczono optimum pH, optimum temperatury i wpływ inhibitorów. Wykreślono krzywe spektralne pero
ksydazy grzybowej i peroksydazy z chrzanu, wykazując podobieństwo obu preparatów.
P E 3 IO M E
ripoBOAHJiMCb nonbiTKM OMMiueHMs nepoKCMfla3bi, coflepłKameiicfl b
(pmibTpaTe TpexHeflenbHbix KynbTyp rpnóa Inontus radiatus. riepBbie ABe cTaflMM OHMmeHMS 3aK/iiOManMCb b AsyKpaTHOH npeunnkiTauHM cynbtpa-
tom aMMom nnocpMnM3OBaHHoro tpM/ibTpaTa. 3aTeM npoBOAnnacb cpnnbT- pauMB Ha KonoHKe Sephadex G 100 (pa3Mepbi kojiohkm 2,5 X 45 cm).
3tom cpmibTpauMeM AocTuraeics 30-KpaiHoe oMnLU,eHne nepoKCHAa3bi no OTHOineHHto k HaHanbHOMy MaTepna/iy. OnpeAenmiM HeKOTopbie cBoncTBa oMMiueHHoro npenapaia tpmÓhom nepoKCHAa3bi h cpaBHmiM nx co cBOMCTBa-
mm nepoKCMAa3bi H3 xpeHa. OnpeAennnH onTHMyM pH, onruMyM reMne- paiypbi u B/iHBHue hhthÓhtopob. BbmepneHbi cneKTpanbHbie KpnBbie rpnó-
hoh nepoKCMAasbi u nepoKCHA^3bi H3 xpeHa, KOTopbie noKa3biBaioT cxoa- ctbo o6ohx npenapaTOB.
Pap. druk. sat. III kl. 80 g Format B5 (70X100) Stron druuk: 9 Annales UMCS, Lublin 1971 Drukarnia Uniwersytecka w Lublinie Zam. nr 12 z dnia 18.1.1971 950 + 50 egz. A-7 Maszynopis otrzymano 18.1.1971 Druk ukończono 25.VIII.71
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